1. Field of the Invention
This invention relates to a reagent for measuring leukocytes and hemoglobin in blood samples.
2. Description of the Related Art
Measurement of hemoglobin concentration and the number of leukocytes in a blood sample is crucial for clinical diagnoses of leukemia, anemia and other hematopathys.
The basic method used for measuring or counting the number of leukocytes is a visual counting method with the aid of a microscope.
However, with the visual counting method, the erythrocytes are hemolyzed by, for example, a Turk solution, while the leukocytes are stained, and the number of the leukocytes is counted one by one on a counting plate which involves a time and labor-consuming operation.
Meanwhile, in counting the number of leukocytes in a blood sample, methods by an automatic blood analyzer are frequently employed. In counting the number of leukocytes by an automatic blood analyzer, an erythrocytolytic agent is added to the blood sample for selective hemolyzation of the erythrocytes in the blood sample to produce a sample for leukocyte measurement in which only leukocytes are left. This sample is passed through narrow paths or orifices provided in a detection section in the automatic blood analyzer, whereby electrical or optical signals are generated at the detection section, the number of these signals being a measure for the number of leukocytes.
Recently, there has also been evolved an apparatus in which, during counting of the number of leukocytes, the leukocytes are classed into granulocytes, monocytes, lymphocytes and so on depending upon the differences in their signal intensities. With the use of this automatic blood analyzer, the leukocytes can be classed and counted for more easily than with the visual counting method.
For hemoglobin measurement, on the other hand, method known as cyanmethemoglobin method is usually employed, according to which hemoglobin in the blood is converted into cyanmethemoglobin (HiCN) under the action of a hemolyzing agent containing potassium ferricyanate and cyanide and the absorbance of the cyanmethemoglobin at a specified wavelength is measured. With the automatic blood analyzer, the cyanmethemoglobin method or the method similar thereto is resorted to since the method can be used directly with the automatic blood analyzer.
However, since toxic cyanides are used with the cyanmethemoglobin method, reagent handling may endanger the operator. In addition, the waste liquor after measurement need be dumped after cyanide decomposition with the use of sodium hypochlorite, for example, by an extremely laborious operation.
For this reason, there is also employed a method, known as an oxyhemoglobin method, whereby erythrocytes are hemolyzed only with nonionic surfactants for converting hemoglobin into oxyhemoglobin (HbO.sub.2), without oxidizing hemoglobin to methemoglobin, for measuring the absorbance at a predetermined wavelength. With the oxyhemoglobin method, since cyanides are not used, there is no risk in handling the reagents, while there is no necessity of performing a troublesome operation of dumping the waste liquor.
However, with the oxyhemoglobin method, a problem is raised that the blood sample with high methemoglobin contents cannot be measured accurately, since methemoglobin then may not be converted to oxyhemoglobin. As an example, Table 1 shows comparison data of the measured results obtained with the Example 1 of the present invention and the oxyhemoglobin method for the cases wherein the methemoglobin contents are changed with the use of control blood. The control blood, which is used as a substance for controlling the analytical accuracy of the automatic blood analyzer, is usually stored in a cooled state, and may exhibit a stable hemoglobin value for a prolonged time. However, on storage at higher than the ambient temperature, hemoglobin in the blood is gradually converted into methemoglobin. Therefore, when the control blood stored at 22.degree. C. is measured by the oxyhemoglobin method, as shown in Table 1, that portion of hemoglobin which has been converted into methemoglobin becomes unable to be measured such that the measured value of hemoglobin becomes gradually lower than the initial value in several days.
As a solution to this problem, a reagent for hemoglobin measurement comprised of dodecyl sodium sulfate or equal amounts of lauryl sodium sulfate (SLS) (an anionic surfactant), and Triton X-100 (a nonionic surfactant), in a neutral buffer (pH, 7.2), is taught by Oshiro et al in Clinical Biochemistry, vol. 15, 83 (1982).
With this method, the hemoglobin concentration in the blood may be measured without being affected by methemoglobin, while there is no necessity of treating the waste liquor because of the absence of cyanide contents.
However, it is not possible with this method to measure leukocytes simultaneously with hemoglobin measurement.
The Japanese Patent Public Disclosure (KOKAI) No. 61-148369 (1986) entitled "CYANIDE-FREE HEMOGLOBIN REAGENT" discloses a reagent for hemoglobin measurement with the aid of an ionic surfactant. However, it is similarly not possible with this reagent to measure the leukocytes simultaneously with the hemoglobin.
As discussed hereinabove, it is not possible with the prior-art technology to measure hemoglobin and leukocytes simultaneously by a cyanide-free reagent or to make a correct measurement of hemoglobin in a blood sample with high methemoglobin contents.